Synthesis, spectroscopic properties and photodynamic activity of Zn(II) phthalocyanine-polymer conjugates as antimicrobial agents

doi:10.1016/j.eurpolymj.2020.109816

European Polymer Journal

Volume 134, 5 July 2020, 109816

https://doi.org/10.1016/j.eurpolymj.2020.109816 Get rights and content

Highlights

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Conjugates of Zn(II) phthalocyanine to chitosan and polyethylenimine were synthetized.
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The basic aliphatic amino groups of polymers can be protonated in a biological medium.
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These conjugates are effective photosensitizers to produce ROS.
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Polyethylenimine conjugate is an efficient photodynamic antimicrobial agent.

Abstract

Conjugates of Zn(II) phthalocyanine to chitosan (CS) and polyethylenimine (PEI) were synthetized through the following stages: first a phthalonitrile substituted by a (carbomethoxy)phenoxy group (Pn 1) was obtained by nucleophilic aromatic substitution reaction, then a AB₃ Zn(II) phthalocyanines (ZnPc 2) was synthesized by the ring expansion reaction of boron(III) subphthalocyanine (SubPc) chloride with Pn 1. After hydrolysis of ZnPc 2, a Zn(II) phthalocyanine bearing a carboxylic acid group (ZnPc 3) was obtained, which was conjugated to CS (CH-ZnPc 4) and PEI (PEI-ZnPc 5) by amide bond. UV–visible absorption and fluorescence spectra presented the characteristic bands of the Zn(II) phthalocyanine in N,N-dimethylformamide (DMF), with appropriate fluorescence quantum yield. Also, the conjugation of Zn(II) phthalocyanine to polymers was confirmed by IR spectra. These conjugates were able to photosensitize singlet molecular oxygen in DMF and aqueous medium. Moreover, they induced the formation of superoxide anion radical in the presence of NADH. The results showed that type II pathway is involved in the photodecomposition of Trp sensitized by these conjugates, although there is also a contribution from the type I mechanism. Photoinactivation of microorganisms was investigated in Candida albicans, Staphylococcus aureus and Escherichia coli and, varying the concentration of CH-ZnPc 4 and PEI-ZnPc 5 and the irradiation times. Both conjugates were efficient in the eradication of S. aureus by PDI, while that PEI-ZnPc 5 was the most effective for photokilling C. albicans. These conjugates were little active to photoinactivation of E. coli. However, the addition of CS allowed to improve the photocytotoxicity towards this Gram-negative bacterium. These results indicate that PEI-ZnPc 5 is an efficient photosensitizer to inactivate S. aureus and C. albicans. In addition, it is capable of killing E. coli cells in the presence of CS.

Graphical abstract

Conjugates of Zn(II) phthalocyanine to chitosan and polyethylenimine are a promising polymeric architecture for use as a broad-spectrum antimicrobial photosensitizer.

Introduction

Antimicrobial resistance is currently a problem that affects worldwide public health [1]. The growing appearance of resistant microbial strains has promoted the development of viable alternatives for the eradication of infectious diseases [2], [3]. In this sense, photodynamic inactivation (PDI) of microorganisms becomes very useful for this application [4], [5]. This promising approach is based on the combination of a photosensitizer (PS), with preferential accumulation in microbial cells, and molecular oxygen (O₂(³Ʃ_g⁻)). After irradiation with visible light, the PS can generate reactive oxygen species (ROS) [6]. These ROS can react with biomolecules inducing a loss of biological functionality and subsequent microbial inactivation [5]. In this approach, two photodynamic mechanisms can occur [5], [7]. The type I mechanism involves the generation of free radicals, while singlet molecular oxygen (O₂(¹Δ_g)) is produced in the type II pathway [7].

At present, there is a wide variety of PSs of great efficiency in PDI, such as porphyrins, chlorins and phthalocyanines, among others [8]. The phthalocyanine derivatives are viable PSs for the photoinactivation of microorganisms due to their physicochemical properties. These compounds have high photostability, high molar extinction coefficient in the region of 650–800 nm and efficient O₂(¹Δ_g) generation [9]. However, phthalocyanines are generally insoluble in organic solvents and present high aggregation in aqueous media as a consequence of their intrinsic planarity of the aromatic system without substituents [10]. These characteristics limit the full application of the PSs, consequently the substitution of the benzene rings in the macrocycle periphery is a promising alternative for the application of the PSs in photodynamic inactivation of microorganisms [11]. In this sense, the asymmetric substitution of the phthalocyanines can be used to improve the limitations of these PSs. Also, the presence of positively charged substituents can increase the solubility of phthalocyanines in biological media, simultaneously improving the photosensitization efficiency to inactivate microorganisms [12], [13], [14]. Another alternative is the formation of conjugates between the PS and positive charge precursor polymers [15], [16], [17], [18], [19], [20]. In this sense, the polysaccharide chitosan (CS) is a natural biopolymer obtained from chitin, which is highly applied in biomedicine and food industries. Also, it presents antibacterial and antifungal activity [21], [22]. On the other hand, polyethylenimine (PEI) is a basic aliphatic polymer that is polycationic due to the presence of primary, secondary and tertiary amino groups [23]. PEI polymers are effective against a variety of Gram-positive and Gram-negative bacteria, including clinical isolates of pathogenic bacteria and bacteria in contaminated water [24]. Both polymers present a numerous amount of free amine groups able to acquire positive charge at physiological pH. This characteristic allows them to interact in aqueous medium with microbial cell membranes. Therefore, these polymers can be used in drug delivery applications, improving solubilization and binding to microorganisms.

In this work, we describe the synthesis of two conjugates formed by a Zn(II) phthalocyanine derivative attached to CS and PEI by amide bonds. The spectroscopic characteristics of these conjugates were determined in solution. Furthermore, the photodynamic capacities of these polymeric materials were evaluated using specific substrates to detect the formation of ROS. In addition, photosensitized decomposition of a biological substrate was analyzed in presence of ROS scavengers to obtain insight about the photodynamic mechanism. The capacity of these conjugates to photoinactivate microorganisms was evaluated in Candida albicans (yeast), Staphylococcus aureus (Gram-positive bacterium) and Escherichia coli (Gram-negative bacterium). The results allow establishing the best conditions for the eradication of microorganisms mediated by these cationic phthalocyanine-polymer conjugates.

Section snippets

4-[4-(carbomethoxy)phenoxy]phthalonitrile (Pn 1)

A solution of 4-nitrophthalonitrile (346 mg, 2.2 mmol), methyl 4-hydroxybenzoate (385 mg, 2.5 mmol) and potassium carbonate (K₂CO₃) (600 mg, 4.3 mmol) in N,N-dimethylformamide (DMF, 10 mL) was stirred for 3 h under argon atmosphere at 30 °C. The reaction mixture was cooled, precipitated with cool water (150 mL) and the solid was washed with methanol (25 mL). The product was purified by flash chromatography column (silica gel, dichloromethane, DCM) yielding 398 mg (65%) of Pn 1. ¹HNMR (CDCl₃) δ

Synthesis

Two conjugates bearing phthalocyanine units attached to CS (CS-ZnPc 4) or PEI (PEI-ZnPc 5) were synthesized from ZnPc 3. This asymmetrically substituted phthalocyanine by a carboxylic acid group was synthesized from a phthalonitrile derivative by a three-step procedure. First, phthalonitrile Pn 1 substituted by a (methoxycarbonyl)phenyl group was prepared through a nucleophilic ipso-nitro substitution reaction of 4-nitrophthalonitrile and methyl 4-hydroxybenzoate in K₂CO₃/DMF (Scheme 1) [33].

Conclusions

In this study, phthalocyanines conjugated with CS and PEI (CH-ZnPc 4 and PEI-ZnPc 5) were synthetized. This requires the formation of a phthalocyanine with AB₃ symmetry (ZnPc 2), which was obtained by the ring expansion reaction of SubPc with the phthalonitrile derivative Pn 1. Hydrolysis of ZnPc 2 yielded a phthalocyanine bearing a carboxylic acid group (ZnPc 3) that was covalently attached to polymers by amide bond. These conjugates can obtain positive charges in biological medium due to the

CRediT authorship contribution statement

Estefanía Baigorria: Conceptualization, Methodology, Writing - original draft. María E. Milanesio: Supervision, Writing - review & editing. Edgardo N. Durantini: Supervision, Writing - review & editing.

Acknowledgments

Authors are grateful to ANPCYT (PICT-2016 0667) and MINCyT Córdoba (PID-2018 36 and GRFT-2018 79) for financial support. M.E.M. and E.N.D. are Scientific Member of CONICET. E.B. thanks to CONICET for the research fellowship.

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations.

Declaration of Competing Interest

The authors declare no competing financial interests.

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Synthesis, spectroscopic properties and photodynamic activity of Zn(II) phthalocyanine-polymer conjugates as antimicrobial agents

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

4-[4-(carbomethoxy)phenoxy]phthalonitrile (Pn 1)

Synthesis

Conclusions

CRediT authorship contribution statement

Acknowledgments

Data availability

Declaration of Competing Interest

J. Infect. Public Health

Drug Resist. Update

Photodiag. Photodyn. Ther.

Eur. J. Med. Chem.

Coord. Chem. Rev.

Coord. Chem. Rev.

Coord. Chem. Rev.

Synth. Met.

Bioorg. Med. Chem.

Bioch. Bioph. Acta

Biomacromolecules

Acta Part A: Mol. Biomol. Spect.

Prog. Polym. Sci.

Photodiag. Photod. Ther.

Reactive Funct. Polym.

Mater. Sci. Eng. C

Progr. Polym. Sci.

Eur. J. Med. Chem.

J. Photochem. Photobiol. B: Biol.

Eur. J. Med. Chem.

Fungal Biol.

Photodiag. Photodyn. Ther.

Synth. Met.

Dyes Pigm.

Synth. Met.